PPARδ inhibition blocks the induction and function of tumor-induced IL-10+ regulatory B cells and enhances cancer immunotherapy

IL-10+ regulatory B cells (Bregs) play a significant role in cancer immunotherapy and their presence is an indicator of negative outcome. We found that PPARδ is significantly upregulated in tumor-induced IL-10+ Bregs with a phenotype of CD19+CD24hiIgDlo/−CD38lo or CD19+CD24hiIgDlo/−CD38hi in both mice and humans, and the level of PPARδ expression was correlated with their potential to produce IL-10 and to inhibit T cell activation. Genetic inactivation of PPARδ in B cells impaired the development and function of IL-10+ B cells, and treatment with PPARδ inhibitor diminished the induction of IL-10+ Bregs by tumor and CD40 engagement. Importantly, immunotherapy with anti-CD40 or anti-PD1 antibody achieved a markedly improved outcome in tumor-bearing mice with PPARδ deficiency in B cells or treated with PPARδ inhibitor. This study shows that PPARδ is required for the development and function of IL-10+ Bregs, providing a new and effective target for selectively blocking Bregs and improving antitumor immunotherapy.

(a) or one-way ANOVA with Tukey's multiple comparisons test (c-e). Shown are data from a representative of two (a-c) or three (d-f) independent experiments.

Supplementary Fig. S2. Combination treatment with immunotherapy and PPARδ inhibitor promotes tumor infiltration of CD8 + T cells and anti-tumor immune memory responses.
(a-d) C57BL/6 mice (n=8-9) were inoculated B16 cells and treated as indicated in Fig 1b. Tumor was dissected at day 24 and analyzed by flow cytometry and IHC. (a) Frequencies (%) of tumor-infiltrating CD3 + T cells and ratios of CD8 + /CD4 + T cells. (b) Representative staining profiles (left) and numbers (per gram tumor tissue) of tumor-infiltrating CD8 + T cells (right). (c) Expression of perforin, granzyme B, CD107a and PD1 in tumor-infiltrating CD8 + T cells. (d) IHC images showing intratumoral distribution of CD8 + TILs (arrows point to T cells). (e) Tumor-free mice were obtained from multiple experiments, in which mice were initially inoculated (s.c.) with B16 melanoma and treated with low-dose FGK plus GSK (n=7) or with high-dose FGK alone (n=6); the overall proportions of tumor-free mice were 32% for the former group and 29% for the latter. These tumorfree mice were rechallenged with i.v. injection of 7×10 5 B16 cells 90 days after initial tumor inoculation, and age matched naïve mice (n=6) were used as the controls. Shown are schematic diagram of experiment setup (top) and Kaplan-Meier curves generated for survival data (bottom). Data are mean ± SEM or representative staining profiles or images. P values were determined by oneway ANOVA with Tukey's multiple comparisons test (a-c) or the log-rank test (e). Shown are data from a representative of two (e) or three independent experiments (a-d).

Supplementary Fig. S3. PPARγ and PPARδ expression in macrophages of tumor-bearing mice is not affected by FGK or GSK.
(a-b) PPARγ-GFP reporter mice (n=3) were inoculated with 7 × 10 5 B16 cells, untreated (control) or treated with T007 (45 nmol; daily for 1 week), and analyzed 1 day after last T007 injection for frequencies, quantifications (left) and levels (MFI; right) of GFP expression in CD11b + F4/80 + cells from spleens (a) and PBMCs (b). (c) Schematic diagram of experiment setup (top). Levels (MFI) of PPARδ expression in dLN CD11b + F4/80 + macrophages of naïve and tumor-bearing mice (at day 17) (bottom, n=3). All data are mean ± SEM. P values were determined by unpaired two-tailed Student's t test (a and b) or one-way ANOVA with Tukey's multiple comparisons test (c). Shown are data from a representative of two (a, b) or three (c) independent experiments. Supplementary Fig. S4. GSK attenuates the ability of tumor-associated B cells to inhibit CD8 T cell function.
C57BL/6 mice (n=8) were inoculated B16 cells and treated as indicated in Fig. 1b. dLNs (inguinal LNs) were harvested at day 17 and assessed for their effects on CD8 T cells. (a-c) CD19 + B cells were sorted from pooled dLNs of the indicated groups and cultured with CD8 T cells or with CD4 + CD25and CD8 + T cells in the presence of anti-CD3/CD28 dynabeads for 3 days, then CD8 T cells were analyzed by flow cytometry for IFNγ production. Shown are representative FACS profiles (a) and percentages of IFNγ + cells in CD8 + T cells that were cultured alone (b) or with CD4 + CD25 -T cells (c). (d-f) CD19 + B cells were sorted from pooled dLNs of the indicated groups and examined for the ability to inhibit perforin and granzyme B production of CD8 + T cells in response to CD3/CD28 stimulation. Shown are representative FACS profiles (d) and percentages of perforin + (e) and granzyme B + (f) cells in CD8 + T cells. All data are mean ± SEM, and P values were determined by one-way ANOVA with Tukey's multiple comparisons test. Shown are data from a representative of two independent experiments. CD19 + CD24 hi IgD lo/-CD38 lo and CD19 + CD24 hi IgD lo/-CD38 hi B cells were sorted from naïve WT and Cd19 Cre/Cre IL-10 f/f (IL-10 KO) mice, and examined for the ability to inhibit T cell activation by measuring IFNγ production of CD4 + CD25 -T cells in response to CD3/CD28 stimulation. Shown are representative FACS profiles (left) and percentages (right) of IFNγ + cells in CD4 + T cells. All data are mean ± SEM, and P values were determined by one-way ANOVA with Tukey's multiple comparisons test. Shown are data from a representative of two independent experiments. Supplementary Fig. S12. Bregs from patients with ovarian or urothelial cancer express higher PPARδ and IL-10 than CD19 + CD24 lo IgD hi B cells.
(a) Representative profiles showing CD24, IgD and CD38 expression on gated CD19 + B cells (left) and percentages of CD19 + CD24 hi IgD lo/-CD38 lo and CD19 + CD24 hi IgD lo/-CD38 hi Bregs (right) in peripheral blood of healthy subjects, ovarian cancer and urothelial cancer patients (n=10 per group). (b-c) Representative FACS profiles (left) and levels (MFI; right) of PPARδ expression in gated CD19 + CD24 hi IgD lo/-CD38 lo , CD19 + CD24 hi IgD lo/-CD38 hi and CD19 + CD24 lo IgD hi B cells from peripheral blood of ovarian cancer (b, n=7) and urothelial cancer patients (c, n=8). Shown are combined data from two independent experiments. (d) Representative profiles showing IL-10 staining (left) and percentages of IL-10 + cells (right) in the indicated B cell populations (n=6 per group). Shown are data from a representative of two independent experiments. All data are mean ± SEM, and P values were determined by one-way ANOVA with Tukey's multiple comparisons test.

Supplementary Fig. S13. PPARδ expression in IL-10 + Bregs is markedly greater than in other lymphocytes and macrophages.
The indicated B cell subsets, CD3 + T cells and F4/80 + macrophages were sorted from naïve mouse spleens and measured for PPARδ expression by western blot. Shown are representative results of two independent experiments.

Supplementary Fig. S14. GSK didn't inhibit function of activated T cells.
CD4 + CD25 -T or CD8 + T cells were sorted from naïve mouse spleens and labeled with CFSE, then stimulated with anti-CD3/CD28 mAbs in the presence or absence of GSK for 3 days. (a, b) Shown are representative FACS profiles (left panels) and cell proliferation rates (right panels) of CD4 + (a) and CD8 + (b) T cells. (c, d) Shown are representative FACS profiles (left panels) and percentages (right panels) of IFNγ + cells in CD4 + (c) or CD8 + (d) T cells. All data are mean ± SEM (n=3), and P values were determined by unpaired two-tailed Student's t test. Shown are data from a representative of three independent experiments.